Characterization of chemical gradients in the collagen gel-visual chemotactic assay

Chemical gradients developing in a collagen gel-visual chemotactic assay (CG-VCA) for PMNs were evaluated by theoretical and experimental methods. First, a video image analysis system was used to establish the diffusion coefficients of bromophenol blue (BPB) through the membrane (D1) and across the collagen gel (D2) in a capillary tube apparatus used only for this purpose. The diffusion coefficients of BPB and the geometry of the CG-VCA system were then used to develop a mathematical model, estimating theoretical gradients in the CG-VCA system. In addition, gradients in the CG-VCA system were characterized experimentally by employing BPB as the source and determining the concentration profiles of BPB in the chemotactic compartment by video image analysis. A relative error of approximately 21% exists between the theoretical and experimental gradients for both 0.5 and 1.0 mM source concentrations of BPB. This favorable comparison demonstrates reliability in predicting chemical gradients for the CG-VCA system. The mathematical model was then used to predict gradients using nanomolar and micromolar concentrations of low molecular weight chemoattractants. Analysis of these specific gradients showed that gradients were steep enough to be detected by PMNs in the collagen gel during the observation period used in previous experiments. The determination of BPB gradients in the CG-VCA system illustrates the utility of this system.